Glucagon-like peptide-1 (GLP-1) was first discovered in 1987 and identified as a glucose-dependent intestinal secreted hormone peptide. GLP-1 peptide transmits signals through G protein-coupled receptors (GPCR), and stimulates β islet cells to secret insulin to inhibit glucagon secretion, gastric emptying and gastric acid secretion and effects other physiological functions.
The exendins are peptides (J. Biol. Chem. 1999, 265, 20259-20262; J. Biol. Chem. 1992, 267, 7402-7405) found in the salivary secretions of venomous lizards the Gila monster and Heloderma horridum. These exendins, as represented by exendin-4, are highly homologous to GLP-1 [7-36]. Previous researches have discovered that the exendins can bind to GLP-1 receptors and exert similar pharmacological effects, such as stimulating insulin secretion, effectively controlling blood sugar levels after a meal, reducing the glycosylation level of hemoglobin and inhibiting gastric emptying. Testing on animals found that long term use of GLP-1 receptor peptides can effectively reduce the resistance to insulin which may lead to the reversion of diabetes mellitus deterioration. Additionally, it has been found that a number of insulinotropic agonists such as GLP-1 and exendin-4 can stimulate regeneration of β islet cells (Nat. Biotech. 2005, 23, 857-861) and ameliorate nonalcoholic fatty livers (Hepatology 2006, 43, 173-181). These discoveries make such peptides a hot area in the studies of diabetes and adiposis. Wu Dengxi and Sun Yukun (Chinese patent: ZL01112856) have modified exendin-4 and obtained a series of exendin-4 analogs with the same functions as the native exendin-4. Recently, a new exendin based drug, exendin-4 (Byetta®), came into the US market. This drug was jointly developed by Amylin and Eli Lilly and requires two injections per day. The drug has drawn attention in the therapeutic field of diabetes and adiposis. However, clinical researches discovered that about 41% of the patients produced antibodies against exendin-4 after 30 weeks of treating with such drug (Diabetes Care. 2004, 27, 2628-2635).
Protein/peptide drugs typically have shortcomings such as a short half life in blood, poor physical and chemical stability, and prone to in vivo degradation by proteases. As a result, multiple injections of such drugs are required every day, causing lots of pain and inconvenience to patients. How to extend the half life of these drugs has puzzled the biotechnology industry for a long time. Presently, no one has found a universally acceptable solution to this problem.
PEG modification technology emerged in the 1970s and was applied in the technology of making protein/peptide drugs. When certain protein/peptides were modified by linear or branched PEG, the modification may have given the following features to the protein/peptide: (1) an improvement in physical and chemical stability; (2) a decrease in immunogenicity; (3) an increase in resistance to protease degradation; (4) an extension of half life in blood due to the increase in PEG molecular weight leading to reduced kidney clearance; and (5) an improvement in drug solubility and cell membrane penetration. According to studies by A. Yang and K. Precourt, exendin-4 is primarily metabolized through kidney clearance. Therefore, they employed a PEG having a molecular weight in the range of 500 to 20,000 Da to modify exendins (Chinese patent: CN1372570A) to reduce the effect of kidney clearance.
However, a main defect of the PEG technology is that the bioactivity of a modified drug generally drops significantly after the modification. Haim Tsubery et. al. employed 9-hydroxymethyl-7-sulfofluorene-N-hydroxysuccinimide (FMS) to activate PEG to be coupled with exendin-4, and then the PEG groups were released from the exendin-4 by in vivo hydrolysis. Thus the bioactivity of exendin-4 was restored. Although this method provides a solution to the problem of low bioactivity due to PEG modification, un-modified exendin-4 was released after in vivo hydrolysis and the problem of immunogenicity resulting from frequent injections has remained unsolved (J. Biol. Chem. 2004, 279, 38118-38124).
Existing defects of known exendins and exendin analogs include short time intervals between doses, the production of antibodies in patients from long-term injection, and the reduction of bioactivity after modification with PEG. These defects make it hard for exendins and exendin analogs to be applied in practice and require the administration of large dosages of exendins or exendin analogs which severely impedes the application of exendin technology.